Antibacterial agents

ABSTRACT

An antibacterial agent comprising one of dicarboxylic compounds or alkali metal salts thereof generally given by the formula 
     
         ROOC(CH.sub.2).sub.n COOR&#39; 
    
     wherein R and R&#39; are a hydrogen or an element of the alkali metal group, respectively, and n is an integer ranging from 11 to 14 inclusive, or a mixture of two or more of said compounds or alkali metal salts thereof, said antibacterial agent being stable in an aqueous solution and a small amount of addition thereof to foods exhibiting an improved antibacterial effect on gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis.

This is a continuation-in-part application of application Ser. No.969,532, filed Dec. 14, 1978, now abandoned, which in turn is adivisional application of application Ser. No. 893,450, filed Apr. 4,1978, now abandoned.

The present invention relates to an antibacterial agent comprising oneof straight-chain saturated dicarboxylic acids or an alkali metal saltsthereof or a mixture of two or more of said dicarboxylic acids or alkalimetal salts thereof and it provides such a non-medical antibacterialagent having an improved antibacterial property.

Hetertofore, since the food contamination with bacteria andmicroorganisms has caused not only a deterioration of taste but also afood putrefaction or poisoning, several kinds of antibacterial agentshave been used to improve the preservability of foods.

Therefore, antibacterial agents such as monoglyceride caprate or sodiumpropionate have been used to prevent the food contamination withbacteria or microorganisms during the course of processing ordistribution thereof. However, since such conventional antibacterialagents do not have a sufficient antibacterial ability, it has beenproposed to add an acid together therewith for maintaining the pH at alower level to reinforce the antibacterial ability.

However, the use of such a method according to the prior art asmentioned above has been limited in that it is effective, by its ownnature, only to acidic foods because of sourness caused by an acid addedthereto. Especially, in the case of monoglyceride caprate, its use islimited from the viewpoint of long-term stability, because an ester bondexisting in the molecule thereof renders it readily susceptible tohydrolysis.

Accordingly, an object of the present invention is to provide anantibacterial agent which has an improved antibacterial ability as wellas safety and in which the aforementioned shortcomings of the prior artantibacterial agents are eliminated.

The inventors have found that a substance having an antibacterialactivity exists in the bark and lignum of Hybrid Aspen (F₁ Hybrid ofJapanese Aspen; Populus sieboldic MIQ, and Canadian Aspen; Big toothAspen Populus grandidentata MICH_(x)) and isolated said substance andidentified to be azelaic acid. Then, the inventors have achieved thepresent invention as a result of a series of intensive studies on theantibacterial activity of straight-chain saturated dicarboxylic acidsand metal salts thereof which are analogs of azelaic acid.

As shown in Tables 1, 2 and 3 and mentioned hereafter, the antibacterialagent according to the present invention comprises one of the compoundsgenerally given by the formula

    ROOC(CH.sub.2).sub.n COOR'

wherein R and R' are a hydrogen of an element of alkali metal group,respectively, and n is an integer ranging from 11 to 14 inclusive, or amixture thereof.

In the meantime, those carboxylic acids having a carbon number of 5 orless give a lower pH in the state of an aqueous solution, while thosehaving a carbon number larger than 18 are not suitable for a practicaluse because of much decreased solubility and weak antibacterial effect.Also, it is preferable to use a sodium salt as the alkali metal saltbecause it can be produced with the highest purity at a lower cost ascompared with potassium salts entailing a higher cost and lower purity.

The antibacterial agent given by the aforementioned formula can bereadily obtained by extraction from the bark or lignum of trees of theaforementioned poplar family or by a simple oxidation of a naturallyexisting material. Also, it is well-known that aliphatic acids such asmonocarboxylic acids are subjected to an omega oxidazation to be turnedinto a dicarboxylic acid by an enzymatic action when taken in a livingbody and finally excreted as a metabolic excreta after playing a certainphysiological role in the living body, and it can be expected that thedicarboxylic acid compounds have an extremely low toxity. In view ofthis, the antibacterial agent according to the present invention hasadvantageous characteristics as a food preservative.

The compounds given by the aforementioned formula has a strongantibacterial ability as mentioned herein before. Especially, thedialkali metal salts having a carbon number of 13 to 16 inclusive havean antibacterial activity on the bacteria such as Staphylococcus aureus,Bacillus subtilis or Escherichia coli 7 to 80 times stronger thanmonoglyceride caprate. Therefore, the added quantity of said compoundsaccording to the present invention can be reduced to 1/7 to 1/80 of theconventional antibacterial agents and the handling thereof can befurther facilitated.

In application of the antibacterial agent according to the presentinvention, one of the compounds given by the aforementioned formula or amixture of two or more of said compounds may be mixed into foods such asbread or soy cake or materials thereof, or may be added thereto incombination with other food additives such as surfactants, withoutsacrificing the antibacterial effect et al. The addition of about 0.005to 0.1% (by weight) thereof can exhibit a sufficient effect as a foodadditive.

Hereinafter, the present invention will be described further in detailby way of preferred embodiments thereof.

PREFERRED EMBODIMENT NO. 1

One loop was inoculated from slant culture of Staphylococcus aureus andBacillus subtilis, respectively, into a liquid culture medium containing1% of glucose, 0.5% of meat juice extract, 0.5% of peptone and 0.3% ofsodium chloride and separately subjected to a pre-culture at 37° C. for24 hours. Then, each 0.1 m of said two resultant cultures from saidpre-culture was separately inoculated into 10 m of each of three culturemedia having the same composition as that used for said pre-culture, towhich the antibacterial agent according to the present invention,conventional monoglyceride caprate and sodium propionate wererespectively added. Then, these three cultures were subjected to a shakeculture in a L-tube at 30° C. for 24 hours. Consequently, theconcentration of the antibacterial agents at which the growth of eachbacterial could be inhibited was determined on the basis of theturbidity of culture due to the growth of bacteria (absorbance at thewave-length of 660 m was measured by means of a spectrophotometer).

Table 1 shows the relationship between antibacterial agents and theminimum concentration thereof activity to inhibit the growth ofbacteria.

                  TABLE 1                                                         ______________________________________                                        Minimum inhibitory concentrations (mg/l)                                                        Types of Bacteria                                                               Staphylococcus                                                                            Bacillus                                      Compounds           aureus      subtilis                                      ______________________________________                                             Pimelic acid       300         350                                            HOOC(CH.sub.2).sub.5 COOH                                                     Disodium pimelate  4000        4000                                           Suberic acid       200         200                                            HOOC(CH.sub.2).sub.6 COOH                                                     Disodium suberate  3000        2500                                           Azelaic acid       150         150                                            HOOC(CH.sub.2).sub.7 COOH                                                                        150         150                                            Disodium azelate   2000        1500                                           Sebacic acid       120         150                                            HOOC(CH.sub.2).sub.8 COOH                                                     Disodium sebacate  1200        500                                            1,9-nonamethylene  75          80                                             dicarboxylic acid                                                             HOOC(CH.sub.2).sub.9 COOH                                                     Disodium 1,9-nonamethylene                                                                       400         200                                            dicarboxylate                                                                 1,10-decamethylene 30          30                                             dicarboxylic acid                                                             HOOC(CH.sub.2).sub.10 COOH                                                    Disodium 1,10-decamethylene                                                                      150         75                                             dicarboxylate                                                                 Brassylic acid     20          30                                             HOOC(CH.sub.2).sub.11 COOH                                                    Disodium brassylate                                                                              50          35                                             1,12-dodecamethylene                                                                             10          25                                             dicarboxylic acid                                                             HOOC(CH.sub.2).sub.12 COOH                                                    Disodium 1,12-dodeca-                                                                            30          30                                             methylene dicarboxylate                                                       1,13-tridecamethylene                                                                            10          10                                             dicarboxylic acid                                                             HOOC(CH.sub.2).sub.13 COOH                                                    Disodium 1,13-trideca-                                                                           30          30                                             methylene dicarboxylate                                                       1,14-tetradecamethylene                                                                          10          15                                             dicarboxylic acid                                                             HOOC(CH.sub.2).sub.14 COOH                                                    Disodium 1,14-tetradeca-                                                                         40          35                                             methylene dicarboxylate                                                       1,15-pentadecamethylene                                                                          30          30                                             dicarboxylic acid                                                             HOOC(CH.sub.2).sub.15 COOH                                                    Disodium 1,15-pentadeca-                                                                         120         100                                            methylene dicarboxylate                                                       1,16-hexadecamethylene                                                                           50          50                                             dicarboxylic acid                                                             HOOC(CH.sub.2).sub.16 COOH                                                    Disodium 1,16-hexadeca-                                                                          300         250                                            methylene dicarboxylate                                                       1,17-heptadecamethylene                                                                          90          110                                            dicarboxylic acid                                                             HOOC(CH.sub.2).sub.17 COOH                                                    Disodium 1,17-heptadeca-                                                                         800         600                                            methylene dicarboxylate                                                       1,18-octadecamethylene                                                                           150         150                                            dicarboxylic acid                                                             HOOC(CH.sub.2).sub.18 COOH                                                    Disodium 1,18-octadeca-                                                                          2500        1500                                           methylene dicarboxylate                                                       1,19-nonadecamethylene                                                                           400         350                                            dicarboxylic acid                                                             HOOC(CH.sub.2).sub.19 COOH                                                    Disodium 1,19-nonadeca-                                                                          4000        4000                                           methylene dicarboxylate                                                  Prior                                                                              Monoglyceride caprate                                                                            500         250                                       art  Sodium propionate  100         2500                                      ______________________________________                                    

PREFERRED EMBODIMENT NO. 2

A predetermined quantity of each of two bacteria, namely, Staphylococcusaureus and Bacillus subtilis, precultured in the same method as thepreferred embodiment No. 1 was inoculated into two different culturemedia (culture media composition being the same as the pre-culture ofthe preferred embodiment No. 1) of pH6 and pH7, respectively. Then, inthe same method as the preferred embodiment No. 1, the concentration ofthe antibacterial agents at which the growth of each bacteria could beinhibited was determined to observe the effect of pH level on saidminimum concentration. The results are summarized in Table 2.

                  TABLE 2                                                         ______________________________________                                        Minimum inhibitory concentrations (mg/l)                                                     Types of bacteria                                                             Staphylococcus                                                                          Bacillus                                                            aureus    subtilis                                             Compounds      pH    6       7     6     7                                    ______________________________________                                             (CH.sub.2).sub.7 (COONa).sub.2                                                                    2000  4000  1500  5000                                    (CH.sub.2).sub.11 (COONa).sub.2                                                                   75    200   35    300                                     (CH.sub.2).sub.12 (COONa).sub.2                                                                   30    150   30    250                                     (CH.sub.2).sub.13 (COONa).sub.2                                                                   30    150   30    250                                Prior                                                                              Sodium propionate   500   1700  250   650                                art  Monoglyceride       1000  2500  2500  6000                                    caprate                                                                  ______________________________________                                    

PREFERRED EMBODIMENT NO. 3

A predetermined quantity of each of two bacteria, namely, Staphylococcusaureus and Bacillus subtilis, pre-cultured in the same method as thepreferred embodiment No. 1 was inoculated into a culture medium (havingthe same composition as the preferred embodiment No. 1) of pH6containing equimolar mixture of the antibacterial agents according tothe present invention. Then in the same manner as the preferredembodiment No. 1, minimum concentration at which the growth of bacteriacould be inhibited was determined. The results are summarized in Table3.

                  TABLE 3                                                         ______________________________________                                        Minimum inhibitory concentrations (mg/l)                                                      Types of bacteria                                                             Staphylococcus                                                                           Bacillus                                           Compounds         aureus       subtilis                                       ______________________________________                                             (CH.sub.2).sub.7 (COOH).sub.2                                                 (CH.sub.2).sub.10 (COONa).sub.2                                                                130          100                                             (CH.sub.2).sub.7 (COOH).sub.2                                                                  70           80                                              (CH.sub.2).sub.11 (COOH).sub.2                                                (CH.sub.2).sub.12 (COOH).sub.2                                                                 25           30                                              (CH.sub.2).sub.13 (COONa).sub.2                                          Prior                                                                              Monoglyceride caprate                                                                          500          250                                        art  Sodium propionate                                                                              1000         2500                                       ______________________________________                                    

PREFERRED EMBODIMENT NO. 4

8 kg of Soybean milk was prepared from soybean 2 kg in the usual way.0.01% of each antibacterial agent according to the present inventionwhich was dissolved or suspended in water was added to the Soybean milk.This mixture was charged into polyethylene tube and 0.3% ofδ-glucuronolactone was added further, which was heated at 90° C. for 40minutes. Then this preparation (Beans curd) was sealed and kept at 20°C. In order to observe the state of putrefication of each soybean curd,Variable cell count was determined every day. The results are summarizedin Table 4.

                                      TABLE 4                                     __________________________________________________________________________    PRESERVABLE EFFECT OF SOYBEAN CURD                                            Variable cell counts                                                          (counts/g)                                                                              Number of Day                                                       Compounds 0  1  2  3     4    5                                               __________________________________________________________________________    (CH.sub.2).sub.11 (COOH).sub.2                                                          (--)                                                                             (--)                                                                             (--)                                                                             (--)  (--) 1.2 × 10.sup.3                            (CH.sub.2).sub.11 (COONa).sub.2                                                         (--)                                                                             (--)                                                                             (--)                                                                             (--)  6.7 × 10.sup.2                                                               8.5 × 10.sup.3                            (CH.sub.2).sub.12 (COOH).sub.2                                                          (--)                                                                             (--)                                                                             (--)                                                                             (--)  (--) 4.7 × 10.sup.3                            (CH.sub.2).sub.12 (COONa).sub.2                                                         (--)                                                                             (--)                                                                             (--)                                                                             (--)  4.5 × 10.sup.2                                                               5.0 × 10.sup.3                            (CH.sub.2).sub.13 (COOH).sub.2                                                          (--)                                                                             (--)                                                                             (--)                                                                             (--)  (--) 5.2 × 10.sup.2                            (CH.sub.2).sub.13 (COONa).sub. 2                                                        (--)                                                                             (--)                                                                             (--)                                                                             (--)  4.1 × 10.sup.2                                                               4.3 × 10.sup.3                            (CH.sub.2).sub.14 (COOH).sub.2                                                          (--)                                                                             (--)                                                                             (--)                                                                             (--)  (--) 8.1 × 10.sup.2                            (CH.sub.2).sub. 14 (COONa).sub.2                                                        (--)                                                                             (--)                                                                             (--)                                                                             (--)  5.5 × 10.sup.2                                                               7.6 × 10.sup.3                            Control   (--)                                                                             (--)                                                                             (--)                                                                             227 × 10.sup.3                                                                6.1 × 10.sup.4                                                               4.0 × 10.sup.5                            __________________________________________________________________________     (--):Variable cell counts is below 300                                   

PREFERRED EMBODIMENT NO. 5

A predetermined quality of each of four microorganisms, namelyEscherichia Coli, Pseudomonas aeruginosa, Penicillium citrinum andCandida albicans, pre-cultured in the same method as the preferredembodiment No. 1 was inoculated into a culture media (having the samecomposition as the preferred embodiment No. 1) containing theantibacterial agents according to the present invention. Then in thesame manner as the preferred embodiment No. 1 minimum concentration atwhich the growth of microorganisms could be inhibited was determined.The results are summarized in Table 5.

                  TABLE 5                                                         ______________________________________                                        MINIMUM INHIBITORY CONCENTRATIONS (mg/l)                                               Microorganisms                                                                                              Can-                                   Compound of pre-                                                                         Escheri- Pseudomonas                                                                              Penicillium                                                                           dida al-                               sent invention                                                                           chia coli                                                                              aeruinosa  citrinum                                                                              bicans                                 ______________________________________                                        (CH.sub.2).sub.11 (COOH).sub.2                                                           1000     (--)       (--)    (--)                                   (CH.sub.2).sub.12 (COOH).sub.2                                                           1000     (--)       (--)    (--)                                   (CH.sub.2).sub.13 (COOH).sub.2                                                           1500     (--)       (--)    (--)                                   (CH.sub.2).sub.14 (COOH).sub.2                                                           2000     (--)       (--)    (--)                                   ______________________________________                                         *(--); No inhibition at 2000 (mg/l)                                      

What is claimed is:
 1. A food containing an antibacterial agent of theformula

    ROOC(CH.sub.2).sub.n COOR'

wherein R and R' are hydrogen or an element of the alkali metal groupand n is an integer ranging from 11 to 14 inclusive in a quantity offrom 0.005 to 0.1% by weight.
 2. The food of claim 1 wherein R is analkali metal selected from the group consisting of sodium and potassium.3. The food of claim 2 wherein R is sodium.
 4. A method for theprevention of bacterial growth in food comprising incorporating intosaid food from 0.005 to 0.01% by weight of an antibacterial agent of thegeneral formula

    ROOC(CH.sub.2).sub.n COOR'

wherein R and R' are hydrogen or an element of the alkali metal groupand n is an integer ranging from 11 to 14 inclusive.
 5. The method ofclaim 4 wherein R is an alkali metal selected from the group consistingof sodium and potassium.
 6. The method of claim 5 wherein R is sodium.